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(11) | EP 0 750 046 A1 |
| (12) | EUROPEAN PATENT APPLICATION |
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| (54) | Ammonia elimination reagent |
| (57) The genus Sulfolobus-derived isocitrate dehydrogenase of a thermo stable type in the ammonia elimination
reagent in solution of the present invention has excellent stability under conditions
of alkaline pHs. By adopting the genus Sulfolobus-derived thermo stable isocitrate dehydrogenase for an assay system of a biological
substance producing ammonia as the reaction product, it can be provided an ammonia
elimination reagent, which can be stored in the state of a solution. |
DETAILED DESCRIPTION OF THE INVENTION
Field of the Invention:
[0001] The present invention relates to an enzymatic assay method of a biological substance producing ammonia as the reaction product, wherein ammonia present in a sample of the biological substance is preliminarily consumed up so as to accurately assay such substance, and also relates to an ammonia elimination reagent for the enzymatic assay method using a thermo stable isocitrate dehydrogenase as a coupling enzyme, the dehydrogenase being preferable for a reagent in solution and having excellent stability at alkaline pHs.
Prior Art:
[0002] Generally, it is routinely conducted the detection of substances in urine, blood serum and the like, including urea, creatinine, creatine, guanine and adenosine as well as the assaying of the activities of various enzymes in relation with these substances. For the detection of such substances and the enzymatic reactions thereof, ammonia is firstly generated, and the resulting ammonia is then converted into glutamic acid via glutamate dehydrogenase (abbreviated as "GLD" hereinafter) to determine at 340 nm the reduction of NAD(P)H via the coupled reaction: the reduction-type nicotine amide adenine dinucleotide (phosphate) [NAD(P)H] → nicotine amide adenine dinucleotide (phosphate) [NAD(P)+].
[0003] Because ammonia is measured as the reaction product in the reaction system, however, ammonia originally present in a sample is also measured and included in the measured value. Therefore, it has been difficult to accurately assay such substances. Thus, ammonia present in a sample should be pretreated with 2-oxoglutaric acid via GLD to be modified into glutamic acid. Because the reaction system ie. ammonia → glutamic acid involves the change of NAD(P)H → NAD(P)+, the generated NAD(P)+ should be again converted into NAD(P)H through the reverse reaction NAD(P)+ → NAD(P)H. Then, a coupled reaction can be induced by isocitrate dehydrogenase using as the substrate isocitric acid, together with metal ions such as magnesium ion or manganese ion. The reaction scheme is shown in Fig.1.
Problems to be Solved by the Invention:
[0004] A great number of diagnostic reagents are likely to be prepared in solution in place of freeze-dry powder in recent years, from the needs of reducing laborious works at a vast number of diagnostic laboratory tests by eliminating the process of dissolving a powdery reagent in a solution each time when the reagent is to be used. However, such reagent in solution has a problem such that assay reagents containing NAD(P)H stable under alkaline conditions are so readily inactivated because of the presence of conventional isocitrate dehydrogenase from yeast that it is very difficult to ensure the long storage life of such reagents. Hence, no satisfactory reagents in solution have been produced yet.
Brief Description of the Drawings:
[0005]
Fig.1 depicts the scheme of the reaction pathway of ammonia elimination;
Fig.2 are graphs depicting the ammonia elimination activities of the iCDH from genus Sulfolobus (A) and of the iCDH from yeast (B), in alkaline pH (pH 8.0); and
Fig.3 are graphs depicting the ammonia elimination activities of the iCDH from genus Sulfolobus (A) and of the iCDH from yeast (B), in alkaline pH (pH 9.5).
Means for Solving the Problems:
[0006] The present inventors have made investigations about isocitrate dehydrogenase in view of such conventional problems. Consequently, the inventors have found that the isocitrate dehydrogenase (referred to as "iCDH" hereinbelow) from acid-fast, thermo stable bacteria of genus Sulfolobus is thermally resistant and highly stable under alkaline conditions. The present inventors have furthermore found that reagents containing NAD(P)H in solution can be stored for a long period of time, by using the iCDH described above in an assay system of a biological substance producing ammonia as the reaction product. Thus, the present invention has been achieved.
[0007] The present invention relates to an ammonia elimination reagent in solution, containing 2-oxoglutaric acid, a reduction-type coenzyme (preferably, NADPH), isocitric acid, glutamate dehydrogenase, and isocitrate dehydrogenase as the reagent components, and being stable under alkaline pH conditions for a long period of time. Additionally, the present invention relates to an assay method of a biological substance producing ammonia as the reaction product, comprising consuming up the ammonia present in a sample of the biological substance, subsequently adding a chelating agent to the sample to terminate the iCDH reaction, and adding simultaneously or thereafter an enzyme producing ammonia as the reaction product to assay the generated ammonia.
[0008] In accordance with the present invention, the bacteria of genus Sulfolobus are acid-fast, thermo stable bacteria; as such bacteria, use may be made of acidocaldarius, brierleyi, solfataricus and other bacterial strains of genus Sulfolobus. The standard bacterial strains include a bacterial strain of Sulfolobus acidocaldarius, listed in catalog under DSM 639 (IFO 15157) and a bacterial strain of Sulfolobus solfataricus, listed in catalog under DSM 1616 (IFO 15331 (the old No. IFO 15158)). Strains DSM 639 and DSM 1616 were deposited under the Budapest Treaty in National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry in Japan under Nos. FERM BP-5567 and FERM BP-5566 respectively, on June 14, 1996.
[0009] The extraction and purification of iCDH relating to the present invention are conducted on the basis of known findings. In accordance with the present invention, the procedure was carried out according to the method described in Japanese Patent Laid-open No. Sho 63-214182 (i.e., JP-A 63-214182 (1988)). Furthermore, the iCDH of the present invention may be recovered by extracting the enzyme from the cultured bacteria of genus Sulfolobus and purifying the enzyme. Otherwise, the iCDH may be a recombinant type, which is generated by inserting a DNA vector having genetic information of the iCDH from genus Sulfolobus into a bacterial strain and recovering the iCDH generated from the transformed bacterial strain. As the host bacteria, then, use may be made of for example, Escherichia coli, yeast, Actinomyces, Bacillus subtilis, and the like.
[0010] In accordance with the present invention, the reduction-type coenzyme includes NADH, NADPH, ATP and the like and the derivatives of these reduction-type coenzymes including thio-NADH (thionicotine amide adenine dinucleotide), thio-NADPH (thionicotine amide adenine dinucleotide phosphate), APADPH (acetylpyridine adenine dinucleotide phosphate) and the like.
[0011] For preparing an ammonia elimination reagent stable in solution, preference is given to a buffer solution having a buffering action around pH 9 to 11 because of the selection of alkaline pHs from the respect of the stability of reduction-type coenzymes in solution, in particular. For example, the most appropriate buffers of various types are listed in the Catalog of Dojin Chemical Research Institute, 19-th edition, 1994; preferable buffers are triethanolamine, TAPS [N-tris(hydroxymethyl)methyl-3-aminopropane sulfonic acid], CHES (N-cyclohexyl-2-aminoethane sulfonic acid), CAPSO (N-cyclohexyl-2-hydroxy-3-aminopropane sulfonic acid), CAPS (N-cyclohexyl-3-aminopropane sulfonic acid) and the like.
[0012] Examples of the ammonia elimination reagent using the iCDH from genus Sulfolobus are listed in the table below to show the reagent components. For the preparation of the ammonia elimination reagent, any buffer having an buffering action at alkaline pHs may satisfactorily be used. In the table, the activity of GLD is expressed as follows; an enzyme activity generating 1 µmol of NADP+ per minute is defined as one unit. If necessary, furthermore, a surfactant such as Briji-35 may be added satisfactorily.
Table 1
| Ammonia elimination reagent composition (composition example) | |
| 2-Oxoglutaric acid | 6.4 mM |
| NADPH | 0.3 mM |
| Potassium isocitrate | 10 mM |
| GLD | 20 to 100 U/ml |
| iCDH | 1 to 10 U/ml |
| MgCl2 | 0.2 mM |
| pH | 9 to 10 (preferably, 9.5) |
[0013] For preparation of the ammonia elimination reagent, the other principal component GLD may be derived from yeast, genus Proteus and genus Bacillus. For preparation of the ammonia elimination reagent, any GLD may be satisfactory, but preference is given particularly to GLD with good stability and higher activity of producing glutamic acid in an alkaline pH.
[0014] In accordance with the present invention, the method for assaying the enzyme activity of iCDH should be conducted as follows, unless otherwise stated.
Assaying temperature: 37°C
Composition of reaction solution :
0.1 M Tris-HCl buffer, pH 8.5
5.0 mM potassium isocitrate
1.0 mM NADP+
5.0 mM MgCl2
Detector : spectrophotometer of Type U-2000, manufactured by Hitachi, Co.Calculation of enzyme activity : The absorbance at 340 nm was measured spectrophotometrically, to determine the absorbance change per minute to calculate the enzyme activity. One unit (1 U) is defined as the activity generating 1 µmol of NADPH/minute.
[0015] For the subsequent culturing and extraction of iCDH, the bacteria of Sulfolobus acidocaldarius DSM 639, FERM BBP-5567 were cultured under aeration in the culture medium of the components shown below in Tables 2 and 3, under the temperature conditions of 80°C for 5 days.
Table 2
The components were sufficiently mixed together, to be then adjusted to pH 3.0 with
sulfuric acid.
| Culture medium composition | |
| Glucose | 1.00 g / 1 liter |
| Yeast extract | 1.00 g |
| Casamino acid | 1.00 g |
| (manufactured by Difco. Co. ) Ammonium sulfate |
1.30 g |
| KH2SO4 | 0.28 g |
| MgSO4 ·7H2O | 0.25 g |
| CaCl2 ·2H2O | 0.07 g |
| FeCl3 ·6H2O | 0.02 g |
| Trace element (× 1000 conc.) | 1.0 ml |
Table 3
Adding dropwise sulfuric acid, the elements were dissolved.| Preparation of trace element (× 1000 conc.) | |
| MnCl2 ·6H2O | 1,800 mg per 1 liter |
| Na2B4O7 ·7H2O | 4,500 mg |
| ZnSO4 ·2H2O | 220 mg |
| CuCl2 ·2H2O | 50 mg |
| NaMoO4 ·2H2O | 30 mg |
| VoSO4 ·XH2O | 30 mg |
| CoSO4 | 10 mg |
[0016] Collecting the bacteria by centrifuge prior to washing, the bacteria were then disrupted by ultrasonication to recover a bacterial extract solution to prepare iCDH. The enzyme activity of the prepared iCDH was assayed by the spectrophotometric method described above. As a result, the bacteria of a wet weight of 1 g corresponded to 1 U enzyme activity at 37°C.
[0017] From the bacteria cultured under the culture conditions described above, iCDH was recovered by the following procedures.
1. Preparation of bacterial extract solution
[0018] Adding an extracting buffer of a 10-fold weight that of the bacteria to prepare a bacterial suspension, the suspension was stirred at ambient temperature for 30 minutes while adjusting the suspension to pH 7.5 using 5N NaOH. The resulting solution of the disrupted bacteria was centrifuged at 8,000 rpm for 20 minutes (using a rotor of Type Therval GS3, manufactured by Dupont Co. Ltd.), and after discarding the precipitate, the supernatant was prepared as a bacterial extract solution.
- Extracting buffer :
- 10 mM Tris-HCl buffer, pH 7.5
1.0 mM EDTA
0.2 mM PMSF
0.02 % sodium azide.
2. Ion exchange chromatography using DEAE-Sepharose CL6B gel
[0019] The bacterial extract solution was diluted 3 fold with pure water, and was then charged into a 5-liter column (DI, 25 cm × 11 cmH) packed with Sepharose CL6B to recover the fraction passing through the ion exchange column as an iCDH-containing fraction.
Column equilibrating solution and washing solution :
10 mM Tris-HCl buffer, pH 7.5
1.0 mM EDTA
0.02 % sodium azide.
Conditions for ion exchange chromatographyFlow : 5 liters/hr
Fractionation : 1 hr/Frac.
3. Dye affinity chromatography using Blue-Sepharose CL6B gel
[0020] The fraction passing through the DEAE-ion exchange column, as the starting material for iCDH purification, was fractionated by affinity chromatography using Blue-Sepharose CL6B. The iCDH bound into the dye affinity column was eluted on 0-0.3 M KCl gradient.
Column equilibrating solution and washing solution :
10 mM Tris-HCl buffer, pH 7.5
1.0 mM EDTA
0.02 % sodium azide.
Conditions for affinity chromatographyColumn size : DI 10 cm × 6 cm H
Gel volume : 500 ml
Flow : 1 liter/hr
- Elution :
- 0 - 0.3 M KCl gradient elution in 10-fold-volume column
- Fractionation :
- 200 ml / Frac.
4. Hydrophobic chromatography using Phenyl-Toyopearl 650 C resin
[0021] Adding ammonium sulfate to the iCDH solution recovered as the eluted fraction to a final concentration of 1.5 M ammonium sulfate, the resulting mixture was then charged into a column packed with a resin Phenyl-Toyopearl 650 C. Washing the column with an excess amount of 1.5 M ammonium sulfate, pH 7.5 to remove unabsorbed substances, the iCDH fraction was collected on 1.5 - 0.5 M ammonium sulfate gradient. Column equilibrating solution and washing solution :
10 mM Tris-HCl buffer, pH 7.5
1.5 M ammonium sulfate
1.0 mM EDTA
0.02 % sodium azide.
Conditions for hydrophobic chromatographyColumn size : DI 5 cm × 6 cm H
Flow : 200 ml/hr
Elution : 1.5 - 0.5 M ammonium sulfate gradient elution in 10-fold-volume column
Fractionation : 20 ml / Frac.
5. Gel filtration using TSK gel G3,000 SW column
[0022] Concentrating the purified iCDH fraction recovered in the above procedure 4 by using an Amicon ultrafiltration membrane, the concentrated fraction was then purified by HPLC on a column packed with TSK gel 3,000 SW to recover a stock solution of the purified enzyme.
- Eluate :
- 0.5 M NaCl
10 mM potassium phosphate buffer, pH 7.0
0.02 % sodium azide
HPLC system : CCPM, manufactured by TOSOH, Co. Ltd.
Column size : TSK gel G3,000 SW column (1 inch)
Flow : 3 ml/minute.
[0023] The general enzymatic performance of the thermo stable iCDH purified from Sulfolobus acidocaldarius DSM 639, FERM BP-5567 by the procedures described above is shown below. Molecular weight : 82 kDa (by gel filtration), dimer.
- Optimum pH :
- 8.0 (when the activity was assayed at 37°C).
- Km for NADP+:
- 66 µM (pH 8.6, 37°C)
42 µM (pH 9.5, 37°C)
48µM (pH 10.0, 37°C)
Example 1
[0024] Using two types of iCDHs, namely commercially available iCDH from yeast (manufactured by Oriental Yeast Industry Co. Ltd.) and the iCDH from genus Sulfolobus (recovered by the purification method described above), comparative tests of the ammonia elimination activities of reagents containing either one of the two types were done at a 10:300 volume ratio of a sample, 100 mM NH4Cl to each of the reagents. The ammonia reagents with various iCDH levels of 0.6, 1, 2 and 6U/ml and with 6U/ml iCDH + 10 mM CyDTA, were prepared. The absorbance change was analyzed over time at 340 nm by an automatic analyzer (Cobas Fara). The compositions of the ammonia elimination reagents and the conditions are shown below.
Conditions of ammonia elimination reagents
Reagent mixing ratio: S:DW:R1:R2=10:5:240:60(in µl).
Sample : 100 mM NH4Cl.
- First reagent (R1) :
- 0 - 6.25 U/ml iCDH added (measured at 37°C)
- Second reagent (R2):
- GLD (150 U/ml measured at 37°C; manufactured by Toyobo, Co. Ltd.)
6.4 mM 2-oxoglutaric acid
0.3 mM NADPH
10 mM potassium isocitrate
0.2 mM MgCl2
0.1 % Briji-35
0.1 M Tris-ethanolamine-HCl buffer, pH 8.0 or 9.0.
- For CyDTA addition :
- 12.5 mM CyDTA added to the first reagent to a final 10 mM concentration.
- Detector :
- Cobas Fara (manufactured by Baxter, Co. Ltd.)
[0025] The ammonia elimination performance of the iCDHs from various origins was analyzed at pH 8.0 and 9.5. The results indicate that the difference in performance is small at alkaline pHs.
Example 2
[0026] An ammonia elimination reagent of the following composition, based on the ammonia elimination reagent composition in Example 1, was subjected to storage and stability tests in alkaline pHs adjusted by a variety of buffers. To a final concentration of 0.2 U/ml, commercially available iCDH from yeast (manufactured by Oriental Yeast Industry Co. Ltd.) and iCDH from genus Sulfolobus (recovered by the purification method described above) were independently added to the reagent composition. The prepared reagents were stored at 4°C and 37°C for 11 days for testing the stability. The results are shown in Table 4.
Composition of ammonia elimination reagent :
[0027]
| 2-oxoglutaric acid | 6.4 mM |
| NADPH | 0.3 mM |
| potassium isocitrate | 10 mM |
| MgCl2 | 0.2 mM |
| Briji-35 | 0.1 %. |
[0028] So as to calculate the residual activity ratio of iCDH under conditions of various pHs and temperatures, the activity immediately after the dissolution of the enzyme iCDH was defined 100 %. The residual activity ratio of the iCDH from yeast was 77 % on day 11 when stored at a higher pH, ie. pH 9.0 and 4°C. No significant decrease of the activity of the iCDH from genus Sulfolobus was observed, which indicates that the enzyme can be stored stably under conditions of alkaline pHs for a long period of time.
Table 4
The pH adjustment of buffers was done at 25°C.| Storage stability of a variety of ammonia elimination reagent solution samples | |||||
| from yeast | from Sulfolobus | ||||
| Sample No. | Buffer type (pH) | 4°C | 37°C | 4°C | 37°C |
| 1 | CHES (pH9.0) | 44.4% | 14.8% | 100.0% | 100.0% |
| 2 | Bicine(pH9.0) | 59.2 | 3.7 | 93.4 | 96.8 |
| 3 | TAPS (pH9.0) | 77.8 | 37.0 | 96.8 | 100.0 |
| 4 | CHES (pH9.5) | 25.9 | 0.0 | 87.5 | 87.5 |
| 5 | CAPSO (pH9.5) | 44.4 | 3.7 | 93.4 | 93.4 |
| 6 | triethanolamine (pH9.5) | 55.6 | 7.4 | 100.0 | 93.4 |
| 7 | CAPS (pH10.0) | 11.1 | 0.0 | 96.8 | 84.3 |
| 8 | CHES (pH10.0) | 11.1 | 0.0 | 93.4 | 81.3 |
| 9 | CAPSO (pH10.0) | 13.0 | 0.0 | 90.6 | 84.4 |
| 10 | triethanolamine (pH10.0) | 40.7 | 1.9 | 93.4 | 84.3 |
Table shows the ratio of the residual iCDH activity to the initial activity of iCDH added, under various storage conditions.
Effect of the invention:
[0029] In accordance with the present invention, it is provided an ammonia elimination reagent which can be readily prepared into a reagent in solution and can be stored in a stable manner for a long period of time, using the iCDH from genus Sulfolobus, the iCDH having greater stability under conditions of alkaline pHs for the ammonia elimination reagent.
1. An ammonia elimination reagent in solution, containing 2-oxoglutaric acid, a reduction-type
coenzyme, isocitric acid, glutamate dehydrogenase and isocitrate dehydrogenase.
2. An ammonia elimination reagent according to claim 1, wherein the isocitrate dehydrogenase
is a thermo stable isocitrate dehydrogenase having excellent stability at alkaline
pHs.
3. An ammonium elimination reagent according to claims 1 and 2, wherein the thermo stable
isocitrate dehydrogenase is derived from genus Sulfolobus.